From 1995 to 2000, a total of 673 Enterococcus faecium and 1,088 Enterococcus faecalis isolates from pigs together with 856 E. faecium isolates from broilers were isolated and tested for susceptibility to four classes of antimicrobial agents used for growth promotion as part of the Danish program of monitoring for antimicrobial resistance. The four antimicrobials were avilamycin, erythromycin, vancomycin, and virginiamycin. Major changes in the use of antimicrobial agents for growth promotion have occurred during the last 6 years in Denmark. The government banned the use of avoparcin in 1995 and of virginiamycin in 1998. Furthermore, the producers have voluntarily stopped all use beginning in 1999. The avoparcin ban in 1995 was followed by a decrease in the occurrence of glycopeptide-resistant E. faecium (GRE) in broilers, from 72.7% in 1995 to 5.8% in 2000. The occurrence of glycopeptide resistance among isolates from pigs remained constant at around 20% from 1995 to 1997. It was shown that, in GRE from pigs, the genes encoding macrolide and glycopeptide resistance were genetically linked and that, following the decrease in the use of tylosin during 1998 and 1999, the occurrence of GRE in pigs decreased to 6.0% in 2000. From 1995 to 1997 the occurrence of erythromycin resistance among E. faecium and E. faecalis isolates from pigs was almost 90%. Use of tylosin decreased considerably during 1998 and 1999, and this decrease was followed by decreases in the occurrence of resistance to 46.7 and 28.1% among E. faecium and E. faecalis isolates from pigs, respectively. Erythromycin resistance among E. faecium isolates from broilers reached a maximum of 76.3% in 1997 but decreased to 12.7% in 2000 concomitantly with more limited use of virginiamycin. Use of virginiamycin increased from 1995 to 1997 and was followed by an increased occurrence of virginiamycin resistance among E. faecium isolates in broilers, from 27.3% in 1995 to 66.2% in 1997. In January 1998 the use of virginiamycin was banned in Denmark, and the occurrence of virginiamycin resistance decreased to 33.9% in 2000. Use of avilamycin increased from 1995 to 1996 and was followed by an increase in avilamycin resistance among E. faecium isolates from broilers, from 63.6% in 1995 to 77.4% in 1996. Since 1996 avilamycin usage has decreased, followed by a decrease in resistance to 4.8% in 2000. Our observations show that it is possible to reduce the occurrence of antimicrobial resistance in a national population of food animals when the selective pressure is removed. Cases in which resistance to vancomycin was linked to resistance to erythromycin were exceptions. In such cases resistance did not decrease until the use of both avoparcin and tylosin was limited.

Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88 Flavobacterium psychrophilum isolates and 134 Yersinia ruckeri isolates) and 313 motile Aeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of F. psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y. ruckeri isolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.

The microbial communities of the ileum and cecum of broiler chickens from a conventional and an organic farm were investigated using conventional culture techniques as well as cloning and sequencing of 16S rRNA genes. Eighty-five percent of the 557 cloned sequences were <97% related to known cultured species. The chicken ileum was dominated by lactobacilli, whereas the cecum harbored a more diverse microbial community. The cecum was dominated by a large group of bacteria with hitherto no close cultured relatives but most closely related to Faecalibacterium prausnitzii. Approximately 49 and 20% of the cecal clones belonged to this cluster in conventional and organic broiler chickens, respectively. We were, however, able to recover a number of these phylotypes by cultivation, and the isolates were shown to be butyric acid producers. The investigation was a descriptive rather than a comparative study of 2 different rearing systems; however, several differences were observed. For instance, Clostridium perfringens was found in significantly higher numbers in the birds from the organic farm compared with the conventional broilers, probably due to the addition of salinomycin to the conventional feed. In the ileum, the abundance of the different Lactobacillus species differed between the 2 broiler types. The culture-based and culture-independent techniques complemented each other well. Strengths and limitations of the different methods are discussed.

Currently Clostridium perfringens-induced necrotic enteritis is a major problem in broiler flocks. In the present study, broilers were inoculated with a combination of Eimeria maxima or overdose coccidial vaccine (one inoculation) with C. perfringens (repeated inoculations). Single C. perfringens, E. maxima or an overdose of live coccidial vaccine inoculations did not result in grossly visible necrotic gut lesions, while combined inoculation resulted in typical necrotic lesions at approximately 4 days after inoculations with C. perfringens in approximately one-half of the inoculated animals. Semi-quantitative histological lesion scoring was done to evaluate gut damage in gut sections of animals in which no gross necrotic lesions were detected. This included scoring of hyperaemia, haemorrhages, the amount of red blood cells and protein precipitate in the lumen, villus fusion and epithelial defects. The villus length and the villus length/crypt depth ratio were also analysed. This approach proved to be discriminative between single E. maxima infection, overdose of coccidial vaccine or C. perfringens inoculations and the non-inoculated control group, and between the double-inoculated and single-inoculated groups. In general, the highest histological scores for gut lesions were observed in the double-inoculated groups, but the single-inoculated groups had higher scores than the control group. It was concluded that oral inoculation of broilers with an overdose of live coccidial vaccine in combination with multiple oral C. perfringens inoculations is a suitable model for necrotic enteritis without inducing mortality of the animals. C. perfringens and Eimeria act synergistically in inducing grossly visible gut damage.

A total of 8.2% of flies caught outside a broiler house in Denmark had the potential to transmit Campylobacter jejuni to chickens, and hundreds of flies per day passed through the ventilation system into the broiler house. Our study suggests that flies may be an important source of Campylobacter infection of broiler flocks in summer.